This invention relates to universal bases and their uses.
Universal bases are extensively used in primers, probes, hybridization, sequencing, cloning and the diagnostic detection of infectious targets. A universal base analogue forms base pairs with each of the natural bases with little discrimination between them (Loakes et al., 1997; Loakes, 2001). Desirable requirements for a universal base include the ability to: a) pair with all natural bases equally in a duplex, b) form a duplex which primes DNA synthesis by a polymerase, c) direct incorporation of the 5′-triphosphate of each of the natural nucleosides opposite it when copied by a polymerase, (d) be a substrate for polymerases as the 5′-triphosphate, e) be recognized by intracellular enzymes such that DNA containing them may be cloned. (Loakes et al., 1997). At present no analogue has been shown to have all these characteristics.
Hypoxanthine functions as a universal pairing base (Graig, 1966). Nearest-neighbor thermodynamics of 2′-deoxyinosine (2-deoxy-β-D-ribofuranosyl-hypoxanthine) pairs in DNA duplexes have been reported (Watkins and SantaLucia, 2005). The general trend in stability was reported as I:C>I:A>I:T≈I:G>I:I. 2′-Deoxyinosine has found use as a universal nucleoside and is far less non-discriminating than nitroazole derivatives (Bergstrom et al, 1997). Tm values vary from 35.4° C. when paired with G to 63.2° C. when paired with C. A universal 2′-deoxyinosine analogue 7-octadiynyl-7-deaza-2′-deoxyinosine has also been disclosed (Ming et al., 2008). The nucleobase of this analogue shows universal binding properties with the four natural bases in a 12-mer oligonucleotide with Tm's that varies from 45° C. for C to 34° C. for G.
Destabilization of a duplex when a natural base is substituted with a universal base is a relatively common occurrence and one of the weaknesses of most universal bases in the art.